Cryptococcus neoformans, 13.5% to varicella-zoster virus, 5.5% to Mycobacterium tuberculosis, and 4.5% to Enterobacteria , and 5% were Post Transplant Lymphoproliferative Disorders. Microorganisms causing M/ME in the general population were infrequent  or absent (Neisseria meningitidis). M/ME caused by Enterobacteria, Staphylococci or filamentous fungi were associated with high and early mortality (50%–70% at 1 year). Graft survival was not associated with the etiology of M/ME, nor was impacted by immunosuppression reduction. Based on these results, we suggest international studies to adapt guidelines in order to improve the diagnosis and the probabilistic treatment of M/ME in SOTRs.Kidney transplant recipients develop atypical infections in their epidemiology, presentation and outcome. Among these, meningitis and meningoencephalitis require urgent and adapted anti-infectious therapy, but published data is scarce in KTRs. The aim of this study was to describe their epidemiology, presentation and outcome, in order to improve their diagnostic and management. We performed a retrospective, multicentric cohort study in 15 French hospitals that included all 199 cases of M/ME in KTRs between 2007 and 2018 . Epidemiology was different from that in the general population: 20% were due to   Meningitis and meningo-encephalitis (M/ME) are potentially life-threatening infections with causes that are well described in immunocompetent hosts -3. AccorHere we describe the epidemiology, presentation, and outcome of M/ME that occurred in KTRs in France between 2007 and 2018, with a 2-year follow-up.KTRs diagnosed with M/ME between 1st January 2007, and 31st December 2018, were identified in fifteen French Academic Hospitals with a kidney transplantation program. Participating centers were the university hospitals from the following French cities: Bordeaux, Lille, Limoges, Lyon, Marseille Montpellier, Nantes, Paris , Rennes, Rouen and Toulouse.The national electronic medical databases of these centers were screened for the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD-10) codes for kidney transplantation and for codes related to CNS infections and their main etiologies see . When av- Adult (≥18 years old) KTRs- Diagnosis of M/ME between 1st January 2007 and 31st December 2018 defined by at least one of these observations:3* CSF pleocytosis over 10 cells/mm* Positive CSF bacterial or mycological cultureC. neoformans [CrAg], Aspergillus, or Streptococcus pneumoniae).* Positive CSF antigen  for a pathogen without hypercellularity- Cerebral abscess without CSF hypercellularity- Subarachnoid hemorrhage- High CSF protein concentration without hypercellularityExclusion criteria:- Medical history, characteristics of the KT, immunosuppressive therapy and anti-infectious prophylaxis protocols.- Clinical, biological, microbiological and radiological presentation at admission for M/ME.i.e., a change of therapeutic class, the discontinuation of a drug, or a decrease of at least 25% of the trough level target.- Therapeutic management, including anti-infectious and surgical treatments but also immunosuppressive treatment modulation, - Clinical and biological outcome after meningitis, including patient and graft survival. The data was collected until the last available follow-up.The following data were collected from the medical charts:3.Meningitis was defined as the presence of cerebrospinal pleocytosis >10 elements/mmEncephalitic features were defined as the presence of one of the following: mental status or cognitive impairment, generalized seizures. Meningo-encephalitis was defined as the association of meningitis and encephalitic features.A CSF was defined as lymphocytic or neutrophilic if its cellularity was made up of >50% of lymphocytes or neutrophils, respectively. It was defined as mixed if the difference between these percentages was below 10%.Causative diagnosis was asserted by an infectious disease specialist based on specific chart review.- Recent KT (<6 months)- A history of immunosuppressive therapy before KT- A history of treatment of a rejection episode between last KT and the M/ME onset- A recent (<2 years before M/ME) cytotoxic chemotherapy treatment- Recent history of hemopathy (<5 years)The “highly immunosuppressed status” was defined as the presence of at least one the following criteria:® v9 .The statistical analysis was performed using GraphPad PRISMThe annual incidence of M/ME was estimated by dividing the number of yearly cases by the number of living KTRs in the 15 participating centers during the same year.2 test as appropriate. In analysis including more than two groups, the data were compared using one-way ANOVA or Kruskal-Wallis test according to their normal or skewed distributions.Quantitative variables are presented as mean ± standard deviation or median  according to their normal or skewed distributions. Qualitative variables are presented as numbers (percentages). Data were compared using the Student’s t-test, the Mann-Whitney test, or the χp-value of < 0.05.Survival analyses were performed using the Kaplan-Meier method. A Log-rank test was performed for the comparison between the groups, with a significant This study was approved by the Paris Public Hospitals (“Assistance Publique-Hôpitaux de Paris”) Office of Data Protection (RGPD) and registered under project number 20181105112928.p = 0.81).Between 2007 and 2018, 199 cases of M/ME, caused by 200 pathogens were diagnosed in 194 patients , with a Patients and Methods). No patients had been treated with eculizumab or any other anti-complement therapy.The characteristics of the patient population are shown in Causes of M/ME were almost homogeneously distributed between Fungi, Bacteria, and Viruses (one-quarter each). The last quarter was divided into parasitic, non-infectious M/ME and M/ME of unknown cause (MUC) .C. neoformans (20%), VZV, 13.5%, Mycobacterium tuberculosis (5.5%), Enterobacterales (4.5%) and filamentous Fungi (4.0%), p = 0.002).Overall, the most frequent microorganisms were Half of the cases occurred within the first 3.4 (IQR [0.91–8.58]) years after KT. The earliest episode was diagnosed on the first day after KT and the latest case was diagnosed 43 years after transplantation.The delay before M/ME onset varied with the group of etiology: viral M/ME occurred after a median delay of 2.5 years (QR 0.7–8.8), fungal M/ME after 2.8 years (IQR 0.7–5.8), bacterial M/ME after 3.5 years (IQR 1.3–8.8), parasitic M/ME after 4.9 years (IQR 2.0–9.8) and M/ME due to non-infectious causes after a median delay of 9.1 years (2.0–11.8).C. neoformans and VZV did not seem to vary in risk in the years following KT.The incidence of M/ME after KT was not linear for all microorganisms and varied according to the cause of MME. M/ME due to CMV and filamentous fungi occurred in the first 3 years after KT , and Grap = 0.0006) and the patients with fungal M/ME more frequently with headaches . Neck stiffness was observed more frequently in bacterial and fungal infections (55% and 52%) than in viral infections .The clinical presentation at admission is described in Almost half of the patients presented clinical and/or encephalographic encephalitis. In more than half of the cases (53.3%), the clinical presentation included extra-CNS manifestations that could facilitate the diagnosis. The extra-neurologic involvements associated with the most frequent causes are summarized in + lymphocyte count before onset was lower in the fungal, bacterial and viral group  than in the parasitic and non-infectious group  and in bacterial and fungal M/ME M/ME compared to the rest of the group (p < 0.0001). We assessed the efficiency of various ROC (receiver operating characteristic) curves of the blood to CSF glucose ratio to isolate specific etiologies. The best performance was achieved with a value of 0.5 in discriminating between bacterial and fungal forms vs. the others  with a sensitivity of 72.5 95% CI [62.6–80.6] and specificity of 64.2 95% CI [54.2–73.1]  meningo-encephalitis without lymphoproliferative disorder.p = 0.79).Eleven patients were diagnosed with tuberculous meningitis, of which only two had a proven diagnostic . Among them, 6/11 (45%) were born outside of Western Europe (mostly in Africa) compared with 58/188 (30%) in the rest of the cohort (The eleven tuberculous meningitis were lymphocytic (100%), with a CSF-to-blood glucose ratio <0.5 in 72% of the cases and a protein concentration in the CSF >1 g/L in 72% of the cases.One-year survival after presumptive treatment was 72%.N. farcinica, N. nova, and N. paucivorans infections). All three of them were brain-space occupying lesions. Two of them were associated with pulmonary lesions that helped the diagnosis. Treatment was medical only, with an association of meropenem and cotrimoxazole or levofloxacin.Three cases of Nocardia infection were included (All seven cases of M/ME occurring within the first year after KT were due to pyogenic bacteria. Listeria (in two patients), ESBL-producing enterobacterales (in two patients), Staphylococci (in two patients) and Entercococcus (one patient) were found as causative pathogens.Klebsiella pneumoniae, three Escherichia coli, one Serratia marcescens, one Enterobacter cloacae, and one Proteus mirabilis . Six- and 12-month mortality was 33%, and 44% respectively.Klebsiella strains isolated from M/ME were retrospectively screened for specific virulence factors .All five cases of Staphylococcus infections were mainly due to S. aureus . All cases of Staphylococcal M/ME were community acquired. No case of pyogenic M/ME initially presented with septic shock.Mycobacteria and Nocardia) were initially all treated with a combination of a third-generation cephalosporin and amoxicillin. Among them, 15 patients (47%) were infected with a bacterium that was not sensitive to this combination. Mortality in the first month was 1/17 (6.7%) for the patients who received an effective treatment and 8/15 (53.3%) for the patients who presented a pathogen that was resistant to the initial therapy, p = 0.01.The thirty-two patients presenting a M/ME due to a rapid growth bacterium .The most frequent fungus was CSF direct examination (India-Ink stain) had a 51% sensitivity, CSF culture 78% (blood 34%) and CSF Cryptococcal Antigen 94% (blood 93%).Aspergillus and Mucorales CNS infections were associated with another localization (sinus and/or lung) in all the patients  lymphocytic meningitis with a higher CSF-to-blood glucose ratio than in the case of other microorganisms  mostly due to EBV-related PTLD (8/10).Intravenous immunoglobulins (5/7), tacrolimus (1/7) and sirolimus were found as possible causes of drug-related meningitis, diagnosed after extensive etiological investigation (exclusion of other possible causes) and after a pharmacovigilance investigation.Staphylococcus aureus M/ME 1 year mortality reached 80%. One-year survival in the group of M/ME of unknown cause was 85%. There was no difference in matter of survival according to the level of immunosuppression.One-year and 10-year post-M/ME patient survival were 74% and 70%, respectively . There wOne-year and 5-year death-censored graft survivals were 82% and 67%, respectively, in the overall population. Mortality was not significantly different between the different etiological groups, nor between the groups of patients undergoing immunosuppression minimization or not .We report the largest multicentric study of M/ME in KTRs, including nearly two hundred cases. We show that M/MEs are evenly caused by a wide array of bacteria, fungi and viruses, some of them characterized by specific clinical and/or biological parameters, most of them usually not found in immunocompetent hosts. We also show that the outcome principally depends on the etiology. For these reasons, we believe that the common guidelines for the treatment of M/ME in immunocompetent patients do not apply to KTRs. Consistently with a recent Swiss national study focusing on CNS in SOTs in general , M/ME isOur study population did appeared as highly immunocompromised. We could not search for risk factors as we chose not to perform a controlled study to favor more numerous inclusions according to our goal.C. neoformans, VZV, Enterobacterales, and Mycobacterium tuberculosis totalize more than half of the cases. There were fewer than 4% of S. pneumoniae and Haemophilus influenzae, and no case of Neisseria meningitidis. Only five patients (2.5%) were diagnosed with L. monocytogenes infection. This strongly contrasts with the epidemiology of M/ME found in the general population  prophylaxis could be one of the reasons for this strikingly different epidemiology . In KTRsWe could not study the role of anti-pneumococcal vaccination in our population as this was optimized and recommended in France during the study period. In addition, its effectiveness remains controversial in SOTRs .M. tuberculosis MTB/RIF Xpert® PCR testing  consistent with what has been found previously . Most of testing .C. neoformans was by far the first cause of M/ME . This is consistent with C. neoformans infection being previously reported as the third-most frequent invasive fungal infection in a large mixed cohort of SOTRs  of immunocompromised hosts and the putative association of cryptococcal infections with chronic kidney failure  simultaneous recurrence in two-thirds of the cases. In VZV-seronegative KT candidates, VZV live-attenuated vaccine represents a very effective prevention and should be considered according to the guidelines , 49.Mycobacterium tuberculosis, GNRs and Staphylococci.Bacterial meningitis was mostly due to Interestingly, and for the first time, GNRs, Enterobacterales, were found as the first cause of pyogenic meningitis. GNR meningitis mostly occurred in the first 5 years after KT and manifested as a critical disease with a very high 1-year mortality. Half of the cases were not secondary to a urinary, blood or digestive infection. There was no case suspect of strongyloidiasis . We hypothesize that gut microbiota alterations in the pre- and post-transplantation setting can play a role. The combination of antibiotic treatment and the direct effects of immunosuppressive drugs on the gut microbiota result in an increase in proteobacteria , 51 assoThe CSF-to-blood glucose ratio did not appear as a reliable tool to identify bacterial M/ME. This may be explained by the frequency of fungal M/ME in immunosuppressed hosts as opposed to the general population. However, this ratio was frequently lower than 0.5 in patients suffering from a bacterial or a fungal M/ME.Staphylococci nor Enterococcus faecium, we suggest a probabilistic therapy with drugs with a good blood barrier penetration, consisting in an association of high-dose parenteral meropenem and linezolid, that would cover all the rapid-growth bacteria not sensitive to the combination recommended in the guidelines. These antibiotics also cover S. pneumoniae, N. meningitidis, L. monocytogenes, and H. influenzae M/ME.Because third-generation cephalosporins and amoxicillin, recommended as a probabilistic therapy in case of M/ME in an immunocompromised population ,6 do notS. pneumoniae and tuberculous meningitis, and should only be used in case of a strong suspicion  as it can be associated with a poorer outcomes in other causes  and some fungal (filamentous fungi) M/MEs were associated with the highest mortality and should be considered as the principal threat in case of M/ME in KTRs.Survival was very heterogenous depending on the diagnosis. Some bacterial , second, the immunosuppression strategies can differ from one country to another and may result in a different epidemiology, and third the absence of a control group prevented us from identifying risk factors.M/ME after KT encompass a wide range of causative diagnoses, mechanisms, and outcomes, of which our study provides a very detailed view. We show that the recommendations for the management of M/ME in the general population cannot be applied to KTRs in France. We believe that further studies should be performed in order to build specific guidelines for the management of M/ME in SOTRs.


Coronavirus disease 2019 (COVID-19) is a systemic disease associated with injury (thinning) of the endothelial glycocalyx (eGC), a protective layer on the vascular endothelium. The aim of this translational study was to investigate the role of the eGC-degrading enzyme heparanase (HPSE), which is known to play a central role in the destruction of the eGC in bacterial sepsis. Excess activity of HPSE in plasma from COVID-19 patients correlated with several markers of eGC damage and perfused boundary region . In a series of translational experiments, we demonstrate that the changes in eGC thickness of cultured cells exposed to COVID-19 serum correlated closely with HPSE activity in concordant plasma samples . Inhibition of HPSE by a nonanticoagulant heparin fragment prevented eGC injury in response to COVID-19 serum, as shown by atomic force microscopy and immunofluorescence imaging. Our results suggest that the protective effect of heparin in COVID-19 may be due to an eGC-protective off-target effect. Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presenting as a systemic disease associated with vascular inflammation and endothelial injury . Using nThe endothelial glycocalyx (eGC) is a delicate gel-like layer coating the luminal surface of the vascular endothelium , 6. It iAt least in bacterial sepsis, the final common pathway of eGC damage appears to be remarkably consistent: the activation and release of the heparan sulfate (HS)-degrading enzyme heparanase (HPSE) , 15. BesSublingual video microscopy and blood sampling were performed in 16 adult PCR-confirmed COVID-19 patients, prospectively enrolled from May to June 2020 at the intensive care units (ICU) of the University Hospital Münster and three local teaching hospitals in a non-consecutive manner. Plasma samples were obtained, centrifuged, and stored at -80°C until analysis. Twelve apparently healthy, age-matched volunteers served as controls Table 1.Real-time intravital microscopy was performed at the bedside with a sidestream dark field (SDF) camera  to visualize the sublingual microvasculature (microvessel diameter 4–25 µm) as reported previously in detail , 17. In The activity of HPSE in EDTA plasma was determined by a heparanase activity assay  according to the manufacturer’s instructions but using an in-house developed HPSE buffer . The dosPlasma levels of Syndecan-1 , hyaluronic acid  and Angiopoietin-2  were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits as described previously , 19.2 enriched environment for at least 3 days until reaching confluence. Intensity analysis was performed with ImageJ software  as previously described  and confocal immunofluorescence microscopy  of HS staining were performed on the human umbilical vein endothelial cell line EA.hy926 essentially as described  , 21. Celescribed , 21. Theescribed , was obtIn vitro data are presented as means and standard error of the mean (SEM) unless otherwise stated. Correlation between PBR and eGC thickness was assessed by Spearman correlation coefficient. To account for both the number of observations from a single experiment and the number of experiments, differences in glycocalyx thickness were tested with a nested analysis of variance and Tukey’s post-hoc test. All tests used were two sided, and statistical significance was set at p<0.05. SPSS  and GraphPad Prism  were used for statistical analyses.Values are presented as absolute values with median and inter-quartile range (IQR). The non-parametric Mann-Whitney U test and the Chi-Square test were used to compare parameters between groups. R=0.6, P<0.001; HS: R=0.66, P<0.001; HA: R=0.8, P<0.001; Syn-1: R=0.81, P<0.001; PBR: R=0.38, P<0.05).COVID-19 patients at the ICU had a median (IQR) Sequential Organ Failure Assessment (SOFA) score of 9 (5 – 15), were predominantly intubated  and showed an in-hospital mortality of 38% (6/16). Compared to healthy subjects, COVID-19 patients showed a significantly higher HPSE activity in plasma Table 1.in vitro . The delta changes of eGC thickness measured by AFM in vitro strongly correlated with concordant PBR values  obtained by videomicroscopy in vivo  to randomly selected sterile-filtered sera  from 6 COVID-19 patients and 5 healthy subjects for 60 minutes. COVID-19 serum, but not serum from healthy subjects, caused a distinctive decrease of eGC thickness R = 0.82, P = 0.003) , interleukin (IL)-6 , and Angpt-2  as well as the eGC marker Syn-1 . Surprisingly, however, HPSE activity did not correlate with the PBR  or plasma HS .In the total cohort, HPSE correlated well with inflammatory mediators, such as C-reactive protein , suggesting that the true HPSE activity in severely ill COVID-19 patients could be even higher than the values actually measured. Adjusting HPSE activity for aPTT  indeed revealed a moderate association with the PBR .To understand this finding in more detail, we attempted to roughly compensate for the presence of heparin [which dose dependently blocks HPSE ] by usinin vitro inhibition experiments with HS isolated from bovine kidney confirmed a dose dependent inhibition of HPSE at concentrations between 0 and 6.25 µg/ml; nearly full inhibition of heparanase was reached at a concentration of 6.25 µg/ml HS  may have partially blocked HPSE activity in COVID-19 patients  and the fund Innovative Medical Research of the University of Münster Medical School (I-RO221907 to AR). The funding sources had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vaccination is the best way to limit the extent of the COVID pandemic. Knowledge of the duration of the immune response will allow the planning of a vaccination protocol. This study aims to validate the complete  immune responses over time in large population groups following the full vaccination of healthcare professionals in real-life conditions and to assess the relationship between antibody levels and T-cell activity in relation to the characteristics of the study group. The samples for the study were obtained from volunteers  on three occasions: before vaccination, T0, then 4–9 weeks after full vaccination (two doses BNT162b2), T1, and 7–9 months after vaccination, T2. The humoral response was investigated by the titre of anti-SARS-CoV-2 IgG antibodies to S1 protein. Assays were performed three times at intervals. The cellular response was assessed in a subgroup of 189 subjects by QuanT-Cell SARS-CoV-2 (IGRA). The assay was performed once. A group of 344 subjects fully vaccinated with the BNT162b2 vaccine were included in the study. The humoral response was observed in 100% of subjects at both 4–7 weeks and 7–9 months, but antibody titres fell by almost 90% in this interval. The cellular response was observed in 94% (177/189) of subjects 7–9 months after the second dose of vaccine. In subjects with a negative cellular response, eight out of 12 smoked. A factor associated with greater immunogenicity of vaccination was past SARS-CoV-2 infection. The administration of full BNT162b2 vaccination (two doses) induces humoral and cellular responses detectable even more than six months after vaccination. Smoking may be a factor associated with impaired cellular response to vaccination. The SARS-CoV-2 virus is the cause of a pandemic with many consequences, not only in terms of health, but also in the economic and social dimensions. Although in recent months the problem of SARS-CoV-2 seems to be easing, another wave of infections is still highly likely.The humoral response following SARS-CoV-2 infection involves the production of antibodies directed against virus particle surface proteins located within the spike and nucleocapsid. The spike glycoprotein contains an S1 subunit containing a receptor-binding domain (RBD) involved in forming the binding of the virus to ACE2 receptor cells of the host. This is how the virus enters the cells. Antibodies neutralise the virus and block its binding to ACE2 receptors. Therefore, the level of antibodies directed to the spike protein appears to be a good indicator of the immune response after virus infection and after vaccination. It is also a biomarker of immunity [T cells limit the spread of infection, remove infected cells, and protect against viral infection . In patiThe first approved vaccine against SARS-CoV-2 was BNT162b2 (Pfizer-BioNTech) based on a novel technique. The vaccine contains genetic material (mRNA) encoding the full-length spike (S) protein and production of ‘vaccine’ antigens takes place in cells of vaccinated person. In clinical trials, protection against infection of more than 95% was provided in the first two months after vaccination, with reductions in hospitalisation and mortality from COVID-19 [First, the persons in the so-called risk group had an opportunity to get vaccinated: healthcare workers and persons over 60. Full vaccination with the BNT162b2 vaccine consists of two doses 30 µg of the product. Full vaccination results in a high synthesis of S protein antibodies (higher than after the disease). Subsequent studies found that antibody levels decline rapidly with time after both vaccination and infection. Increased susceptibility to infection was also observed in the vaccinated group and those after infection. The kinetics of antibody titers after vaccination may be of practical importance in developing vaccination programmes. The cellular immunogenicity of vaccines is also of interest to researchers. The role of T cells in response to vaccination is not well understood but is thought to have a significant impact on vaccine efficacy and protection against infection . CurrentThis study aims to validate the complex  immune response over time in large population groups following mRNA vaccination (BNT162b2) of healthcare workers in real-life conditions and to provide an assessment of the relationship between antibody levels and T-cell activity in relation to the characteristics of the study group, including the identification of factors associated with vaccine immunogenicity and the persistence of long-term  responses to vaccination.n = 460 + 272). Of these, 344 received the full vaccination and had blood drawn three times. They were vaccinated with Pfizer-BioNTech’s BNT162b2. The first dose was administered in December 2020/January/February 2021 (25 December 2020–10 February 2021), and the second dose January/February/March (17 January–8 March 2021). The median interval between vaccinations was 21 days (IQR: 21–21). The study, which began in November 2020, involved 732 medical personnel from two hospitals , and before the third dose 25 August–29 September 2021 . The healthcare workers group was divided according to the nature of their work into the following categories: doctors, nurses/paramedics, physiotherapists, care managers, room attendants, administrative staff, laboratory staff.n = 189) in the third blood draw with the Quan-T-Cell SARS-CoV-2 IGRA assay from Euroimmun.The kinetics of the response to vaccination was examined by the titres of anti-SARS-CoV-2 IgG antibodies against the S1 protein (S1 subunit of the S protein) located within the virus spike in blood samples collected on three occasions. Antibody levels before vaccination were indicated as IgGT0, 4–9 weeks after full vaccination as IgGT1, and approximately 7–9 months after vaccination (before the booster dose) as IgGT2. The cellular response was tested once in a group of employees of one hospital , which allows precise quantitative testing of IgG class neutralising antibodies directed against the S1 protein of SARS-CoV-2 virus . The results are given in standardised international units: BAU/mL (BAU = Binding Antibody Unit). The test is used to assess the immune response to vaccination and after infection with the SARS-CoV-2 virus. Tests were performed following the manufacturer’s instructions. As per manufactured recommendations, antibody levels above 35.2 BAU are considered a positive result. Cellular immune response measurements were performed with a EUROIMMUN QuanT-Cell SARS-CoV-2 (IGRA) assay, which is used to assess the activity of T cells stimulated by a pathogen presence. The T cells present in the blood of a person who has been in contact with the virus or received vaccination can recognise the virus antigens. The test involves the in vitro stimulation of T cells with the S1 subunit of the SARS-CoV-2 virus spike protein. Activated T cells synthesise and release IFN-γ. Quantitative assessment of secreted IFN-γ is performed by ELISA. Tests were performed following the manufacturer’s instructions. As per manufactured recommendations, antibody levels above 200 mIU/mL are considered a positive result. Data on health status and lifestyle are based on surveys conducted among the subjects.Adverse effects after the first and second dose of vaccination were assessed in separate surveys, including the occurrence of local  and general  reactions, and grading of severity on a scale of 0: none, 1: mild, 2: moderate 3: severe, 4: very severe, based on the Food and Drug Administration’s guidance for toxicity grading scales for vaccines .This study received approval from an independent ethics committee (No. KB 634/2020) and fulfilled the ethical guidelines of the Declaration of Helsinki. All study participants gave written informed consent before enrolment.2 and Fisher’s exact test. Evaluation of data normality was performed using the Shapiro–Wilk test. Non-normally distributed continuous variables were compared using the Mann–Whitney–Wilcoxon test. Multiple comparisons were made with the Kruskal–Wallis test, post-hoc Dunn test, and multivariate ANOVA (MANOVA) and logistic regression. Natural logarithms of applied values were calculated and compared using parametric tests (t-test and ANOVA) to examine immunoglobulin level changes. p-value was considered statistically significant.Statistical analysis was performed using R 3.6.1 of the R Project for Statistical Computing, Vienna, Austria. Categorical variables were presented as frequencies with percentages, whereas median with interquartile range (IQR) or mean with standard deviation (SD) were used to describe continuous variables. Categorical variables were compared using χThe study population consisted of 344 staff members of two hospitals who received the full vaccination and from whom three blood samples were collected at the following intervals: before vaccination, 4–9 weeks after vaccination, and 7–9 months after vaccination. n = 281). Chronic diseases were reported in 108 out of 344 (31%) participants.The mean age of the subjects was 49 ± 11 years, and the median was 50. The youngest subject was 22, and the oldest was 72 . Women an = 153); non-surgeons, 21% (n = 71); administrative staff, 10% (n = 33); laboratory staff, 15% (n = 51); surgeons, 5% (n = 17); in addition to physiotherapists, ward attendants, and stretcher-bearers.Amongst the subjects, the largest group were nurses/care managers/paramedics, 44%  for IgGT0 antibodies before vaccination was reported in 119 subjects. In the study group, 191 out of 344 (55.5%) subjects did not present past SARS-CoV-2 infection.In the assay after full vaccination , all subjects had positive results for anti-SARS-CoV-2 IgGT1 antibodies according to the manufacturer’s reference value (35.2 BAU/mL). The median antibody level after full IgGT1 vaccination was 2511.0 BAU/mL (interquartile range-IQR: 1518.00–4455.75), min. 90.57, max. 18,665. In a blood sample taken before the third dose (7–9 months after the second one), the median anti-SARS-CoV-2 IgGT2 antibody level was 261.8 BAU/mL (IQR: 157.66–732.75), min. 35.23, max. 8960. All results were also positive in this assay, but the values were lower by more than 9.5 times. The lowest values were close to the cut-off point.As expected, the titre of antibodies decreased in time from the second dose .The level of serological response to IgGT1, IgGT2 vaccination was not correlated with age, gender, BMI, blood group, chronic diseases, smoking, physical activity, or influenza vaccination.The cellular response was tested in 189 subjects, median: 1223.1 mIU/mL (IQR 487.33–3342.61), min. 0.5; max. 65,617, and 12 subjects had negative results based on the manufacturer’s standard (<200 mIU/mL).p = 0.02). In women, the median QuanT-Cell level was 1120.52 mIU/mL (IQR 540.59–2725.39), while in men, 2677.58 mIU/mL (IQR 703.96–5649.12).There was a statistically significant difference in cellular response (based on QuanT-Cell result) according to gender (p = 0.062—borderline result). Overweight subjects had a higher QuanT-Cell response rate, with a median of 1486.09 mIU/mL (IQR 709.23–4444.45), while those with a normal BMI had 1161.83 mIU/mL (IQR 357.39–3036.37).There was a positive trend between the level of cellular response and BMI (p = 0.0018), median QuanTcell: 1775.81 mIU/mL (IQR: 786.2–5474.88) vs. 951.8 mIU/mL (IQR 381–2534.95) in those with and without past SARS-CoV-2 infection, respectively. There was no significant difference in cellular response in the SARS-CoV-2 infection severity analysis.Higher responses in the T-cell activity test were reported in persons who had an infection . The QuanTCell response decreases with time after full vaccination, as shown in the QuantCell values depending on the time since the second vaccination. p < 0.001) and IgGT2   .There were no statistically significant differences between the positive and negative QuantT-Cell groups in terms of age, gender, BMI, chronic diseases, blood group, and VAE. p = 0.014).It was shown that eight out of 12 persons (67%) with a negative QuanT-Cell result were smokers (p < 0.05). The value in persons with a history of infection before vaccination was almost twice as high.Subjects without past infection had significantly lower anti-SARS-CoV2 IgGT1 antibody titres compared to those with confirmed infections. Median IgGT1 antibody titres were 1922 BAU/mL (IQR 1193–3105) vs. 3597 BAU/mL (IQR 2151–5557.75), respectively (p < 0.05), which was 3.7 times higher.For assays at 7–9 months, the statistically significant differences in antiSARS-CoV-2 IgGT2 antibody levels persisted. The median for those without a past infection was 178.02 BAU/mL (IQR 110.76–283.99), while those with confirmed infection, 663 BAU/mL (IQR 295.3–1303.25)  and IgGT1 and IgGT2 antibody titres .In a study of the relationship of the decrease in antibody titre with IgGT1 to IgGT2 values, no association was found with age, gender, BMI, and chronic diseases.Between the second and third collection, median days 182 , there was a more than nine-fold decrease in antibody titres (median anti-SARS-CoV-2 IgGT1: 2511.0 vs. median anti-SARS-CoV-2 IgGT2: 261.8). There was no association of antibody decline with age, gender, BMI, blood group, chronic diseases, smoking, physical activity, or VAEs. Based on statistical analysis, there was a significantly lower decrease in titers of antibodies in persons with past SARS-CoV-2 infection, but no correlation with the severity of the infection course. In those without past infection, the median level of the logarithmic antibody difference was −2.31 (−2.74 to −1.94), and in those with past infection, it was −1.69 (−2.15 to −1.30).In the analysed group, vaccine adverse events (VAEs) after the first dose of COVID-19 vaccine were observed in 95% of subjects (327 out of 344), while after the second dose in 89% (309 out of 344). The predominant adverse events (regardless of severity) were local pain , oedema , and redness at the injection site  . The most commonly reported systemic symptoms were fatigue , headache , and myalgia   .After the first dose of the vaccine, mild (0–5 points), moderate (6–10 points) or severe VAEs (>10 points) were observed in 67%, 17%, and 16% of subjects, respectively, and after the second dose of the vaccine in 52%, 24%, and 24% of subjects, respectively. The median severity of VAEs after the first dose of vaccine was 3.00 (IQR: 2.00–7.00). After the second dose of vaccine, the median was 5.00 (IQR: 2.00–10.00). There were no severe VAE cases complicated by anaphylactic shock in the analysed group, requiring hospitalisation or leading to death. p = 0.34) and anti-SARS-CoV-2 IgGT2 (p = 0.206) antibody titres.There was no association between the VAE severity after the first dose of COVID-19 vaccination and anti-SARS-CoV-2 IgGT1 (p = 0.015). Subjects with moderate and severe VAEs had a greater humoral response (higher antibody titres) compared to subjects with minor VAEs—median antibody titres, respectively: 2989.0 BAU/mL (IQR: 1518.00–5008.65) (p = 0.035), 2805.50 BAU/mL (IQR: 1590.25–4870.50) (p = 0.023), 2163.00 BAU/mL (IQR: 1424.00–3911.38)—for VAEs of 6–10 points, >10 points and 0–5 points.There was a correlation between the VAE severity after the second dose of vaccine and the anti-SARS-CoV-2 IgGT1 antibody titre ; median titre for VAEs > 10 points 348.32 BAU/mL (IQR: 162.22–890.75) vs. VAEs of 0–5 points 239.0 BAU/mL (IQR: 147.7–3651.00), respectively.Subjects with severe VAE symptoms after the second dose of vaccine, compared to subjects with mild VAEs, had higher titres of anti-SARS-CoV-2 IgGT2 antibodies (p < 0.001). Furthermore, a correlation was observed between muscle pain and IgGT1 and IgGT2 titres (p < 0.01).Analysing the association of specific VAEs with vaccine response, a statistically significant correlation was found between the severity of fever and chills after the first and second vaccine doses and IgGT1 and IgGT2 antibody titres .There was no difference between the VAE severity after the first and second vaccine doses and the decrease in antibody titres (logarithmic lnIgGT2–InIgGT1 antibody difference)  for anti-SARS-CoV-2 IgG antibodies (EUROIMMUN QuantiVac ELISA) in both the first sample collected at 4–9 weeks (anti-SARS-CoV-2 IgGT1) and the second one at 7–9 months after full vaccination (anti-SARS-CoV-2 IgGT2). We demonstrated that the humoral response after vaccination was maintained in vaccinated hospital staff more than six months later (7–9 months after the second dose). The persistence of IgG antibodies for months after full vaccination in a significant proportion of individuals in selected populations of medical personnel has also been described in previous papers ,13,14,15Convincing data show a poorer humoral response in men, obese individuals and the elderly ,15. HoweA crucial factor affecting anti-SARS-Cov-2 IgG antibody titre is past SARS-CoV2 infection ,18,19,20The phenomenon of a stronger response to vaccination in individuals with past infection may be explained, among other things, by the fact that recovery from infection may induce an immune response directed against various antigens of the virus and not just the spike protein as in the case of vaccination. Ferrari et al. showed that past SARS-Cov-2 infection provides an immunological memory that persists for many months. The administration of one vaccination dose in persons with COVID history acts as a ‘booster’ and causes a stronger humoral immune response (an antibody titre approximately 10 times higher than in persons without COVID history). It was shown that even persons with low antibody titres after being ill are able to mount a strong immune response after a single dose of the vaccine . HoweverAli Ahmad et al. examined the humoral response in more than 1000 persons vaccinated against SARS-CoV-2 by assessing the levels of anti-S1 antibodies in IgG, IgM and IgA and neutralising antibodies . CompariIn the context of the influence of past infection on vaccine immunogenicity, the relationship with the disease course should be emphasised. A positive correlation of the symptom severity of SARS-CoV-2 infection with the antibody titre was also demonstrated by Wolszczak-Biedrzycka . AlthougIn our group, we did not observe any association of vaccination response with chronic diseases. Other authors are yet to describe an association between thyroid disease and immune response after vaccination or an association between hyperglycaemia, diabetes, and the kinetics and persistence of neutralising antibodies against the SARS-CoV-2 spike protein .As expected, we observed a decrease in antibody titres over time. Like Olariu et al., we noted a reduction in antibodies by as much as 90% (median anty-SARS-CoV-2 IgGT1 antibodies: 2511.0 vs. anty-SARS-CoV-2 IgGT2 antibodies 261.8) over 7–9 months . On the Cellular immunological response to vaccination after 7–9 months was achieved in more than 93% of the study group. Male gender was associated with higher levels of cellular response. In addition, higher Quan-T-Cell values were noted in overweight subjects (borderline results). Choi evaluated the cellular response to a vector vaccine, which generally shows greater cellular immunogenicity than mRNA vaccination . Similarp = 0.035) and severe VAEs (p = 0.023) after the second dose of the vaccine had higher antibody titres than those with minor VAEs in short- and long-term follow-up. However, only borderline significance was achieved in assessing antibody titres 7–9 months after the second dose (p = 0.6). However, we did not confirm the association of VAEs with the cellular response. The relationship of VAE severity to response to vaccination remains unclear and requires further study. However, our results suggest that reactogenicity may be an indicator of immune response to vaccination.Also of interest is the association of VAEs with the immune response to vaccination. VAEs are frequently reported in COVID-19 vaccinated individuals, regardless of the type of vaccine, but in most cases, they involve local adverse reactions of mild severity and short duration . AccordiThis analysis of the immune response to vaccination also has a practical context. There were six confirmed cases of SARS-CoV-2 infection in the study group in the period 7–9 months after vaccination. Three occurred after the first dose and three more after full vaccination. The course of the infections was mild. Tré-Hardy found infection in only one out of 201 medical personnel at 6-month follow-up; Ferrari reported the same in 12 out of 1054 persons . ‘BreaktHowever, the relationship between antibody concentration and the infection rate is straightforward. Analyses of vaccine efficacy data in large populations show that efficacy decreases with time after vaccination, even when virus variability is considered . The effThe decrease of the antibody titre was the reason for introducing the third vaccination dose. The determination of protective antibody levels against infection/severe disease is still the subject of ongoing research ,8,10,36.The decreasing humoral response to vaccination raises the question of the level of cellular response, which in our study was assessed in parallel with the determination of anti-SARS-CoV-2 IgG3 antibodies. Cellular and humoral responses show a moderate positive correlation. However, especially in the graphical representation, it can be seen that there are those with a high cellular and low humoral response among the vaccinated individuals and the opposite with a high humoral and low cellular response. For the time being, it is difficult to explain these discrepancies, which researchers still study ,37.Our study was performed in a day-to-day practice setting in a population of hospital staff; due to the ongoing pandemic, coordinated blood collection and vaccination was possible. In addition, health and lifestyle data were obtained from the subjects. However, it cannot be excluded that individuals with poorer health status, multiple chronic diseases, autoinflammatory diseases, and immunosuppressive treatment postponed the vaccination, and our group was therefore not fully representative. In Poland, vaccination has been mandatory for medical personnel since 1 March 2022. Due to the highly dynamic nature of humoral and cellular responses over time, the exact interval between vaccination administration and sample collection may have been important in the statistical analysis of the relationships studied. Data on health status, vaccination, SARS-CoV-2 infection, and post-vaccination symptoms were derived from questionnaires completed by patients. Medications taken were not analysed. This may have influenced the underestimation of reported comorbidities with a significant impact on the response to vaccination ,39,40.The study results indicate that mRNA vaccination BNT162b2 is safe and effective until about 7–9 months.Understanding the immune response to COVID-19 vaccination is key to developing effective preventive measures during this pandemic. Comprehensive immune response studies derived from real-life practice in large cohorts can make an important contribution to our knowledge. There are few such data in the literature to date. Identifying critical factors that significantly determine the response to vaccination will enable the introduction of an appropriate strategy to implement the vaccination programme.


Amaranth is one of the synthetic azo colorants used to improve the appearance and to increase the appeal of some foods and soft drinks. The excessive consumption of amaranth can be associated with health side effects, emphasizing the need to monitor this food dye. Accordingly, the present study aimed to introduce an electrochemical sensor of glassy carbon electrode (GCE) modified with N-doped reduced graphene oxide (N-rGO), N-rGO/GCE, to detect the amaranth sensitively and rapidly. Several electrochemical techniques such as differential pulse voltammetry (DPV), linear sweep voltammetry (LSV), chronoamperometry (CHA), and cyclic voltammetry (CV) are exploited for the evaluation of the efficiency of the developed electrode for the detection of amaranth. We found that N-rGO/GCE enhanced amaranth oxidation, thus significantly elevating the current signal. Amaranth showed that calibration curves ranged from 0.1 to 600.0 µM, and the limit of detection (LOD) (S/N = 3) was 0.03 μM. Finally, the developed sensor was effectively applied for real samples  with acceptable recovery values from 96.0 to 104.3%. In factAmaranth has been detected by different techniques, including thin layer chromatography (TCL) , high-peRecently, nanomaterials have been widely used in various fields due to their exceptional properties ,37,38,39More recently, semiconductor-like properties can be achieved by chemically doped graphene, and charge conductivity and chemical reactivity can be enhanced by N-/P-doped graphene . NitrogeThus, in this work, we developed a new electrochemical sensor to detect amaranth, in which a GCE was modified with N-rGO. The proposed method has advantages such as high linear dynamic range, high sensitivity, and a low limit of detection. Furthermore, this sensor successfully detected amaranth in real samples.All electrochemical determinations were recorded by means of an Autolab potentiostat/galvanostat . We exploited a single component three-electrode cell with a platinum auxiliary electrode and an Ag/AgCl (3 M KCl) reference electrode to perform the measurements. We used N-rGO/GCE as the working electrode while a Metrohm 827 pH meter  was exploited to control the pH of the solutions. Analytical grade chemicals such as amaranth and other reagents were from Sigma-Aldrich and used as received.3·H2O (wt. 30%)). Next, urea (6.0 gr) was added and ultrasonicated for three hours. Then, the prepared solution was kept in an autoclave reactor at 180 °C for 12 h. After the autoclave reached ambient temperatures (25 °C), the samples were separated by centrifuge, washed several times by using deionized water, and its pH was adjusted to neutral. At last, the products (N-rGO sheets) were freeze-dried.The N-rGO sheets were prepared by a simple hydrothermal method. Firstly, exfoliated GO (100 mg) was dispersed by ultrasonication in deionized water (100 mL), and the pH of the above solution was adjusted to pH = 10 by dropwise adding ammonia solution  in deionized water was prepared, and 4 μL of the N-rGO suspension was drop casted onto the GCE surface and allowed to dry completely in ambient conditions.The synthesized N-rGO sheets were used for the modification of a −1), C=O (1720 cm−1), C=C (1577 cm−1), phenolic C-O (1381 cm−1), and epoxy C-O-C (1038 cm−1). These index peaks indicate that there are several oxygen-containing functional groups  on the surface of GO. Moreover, for N-rGO, the absence of absorption peaks of oxygen groups indicated that GO has been effectively reduced during the hydrothermal treatment. In addition, there are two peaks at 1562 cm−1 and 1184 cm−1, which can be attributed to C=N (sometimes C=C and C=N bonds can stretch at the same wavelength) and C-N. This result confirmed that Gr was successfully N-doped. The FT-IR spectrum of the as-prepared sample was consistent with previous reports [A comparison of the FT-IR spectrum of GO and N-rGO is given in  reports ,61,62.2O molecules and various oxygen groups on its surface. In comparison, the XRD pattern of N-rGO confirmed the high reduction degree, as depicted in GO and N-rGO were characterized by XRD . The XRDThe FE-SEM image of N-rGO, presented in  showed tWe exploited energy dispersive X-ray spectroscopy (EDS) techniques for the analysis of elemental compositions . The EDSThe obtained results indicate that amaranth electro-oxidation occurred through electron and proton exchange. Furthermore, the optimization of the pH value in detecting the analyte was necessary. We used the DPV technique to explore the electrochemical response of amaranth on the N-rGO/GCE surface in phosphate buffer solution  for pH ranging from 2 to 9. What is noteworthy is that the amaranth electro-oxidation on the N-rGO/GCE surface, in neutral conditions, was higher than in alkaline or acidic medium. Accordingly, the value of 7.0 was chosen as the optimal pH for amaranth electro-oxidation on the N-rGO/GCE’s surface.The electrochemical performance of N-rGO/GCE in comparison with bare GCE was studied by the CV technique at exposure to 100.0 μM of amaranth at 50 mV/s in PBS (0.1 M). The CVs of all as-fabricated electrodes in this study can be observed in We investigated the effect of the scan rate on the amaranth oxidation peak to analyze the electrode processes. We recorded the linear sweep voltammograms (LSVs) for 100.0 μM of amaranth in PBS (0.1 M) at different scan rates. We found a linear dependence of the anodic peak currents on the square root of the scan rate . Such a −1/2 plots have been utilized for the optimal fits of different amaranth contents in PBS (0.1 M). Using various amaranth contents on N-rGO/GCE, we performed chronoamperometric measurements with the working electrode at the potential of 0.6 V. The slopes of the obtained straight lines versus amaranth contents were drawn to exploit the Cottrell equation below:2/s) is the diffusion coefficient and Cb (mol/cm3) indicates the bulk content. There was a linearity for the I plots against t−1/2 under diffusion control over various amaranth contents. We used the slope vs. amaranth content plot  are shown in The ability of N-rGO/GCE for sensor applications in the detection of amaranth was determined in real specimens of tap water, apple juice, and orange juice according to the standard addition method, as seen in In this study, we developed a novel sensitive electrochemical sensor, based on N-rGO sheets, for the detection of amaranth. The proposed sensor exhibits remarkable electrocatalytic activity and excellent sensitivity toward amaranth detection with a linear response over a wide concentration range (0.1–600.0 µM) and a low detection limit (0.03 µM). Moreover, the applicability of N-rGO modified GCE was tested in real samples with good accuracy and satisfying recovery.


Conclusion: WG did not significantly increase bile acid excretion in feces or decrease serum levels of total cholesterol. Moreover, WGP and WG both presented significant antioxidant activity in vivo (p < 0.05) and caused a significant reduction in cholesterol micellar solubility in vitro (p < 0.001). Therefore, WGP may effectively prevent hyperlipidemia and its complications as WGP treatment enhanced antioxidant activity, decreased the concentration of serum lipids and improved the activity of enzymes involved in cholesterol metabolism.Background: So far, no articles have discussed the hypolipidemic effect of wheat germ protein in in vivo experiments. Objective: In this study, we investigated the effects of wheat germ protein  and wheat germ  on cholesterol metabolism, antioxidant activities, and serum and hepatic lipids in rats fed a high-fat diet through gavage. Methodology: We used 4-week-old male Wistar 20 rats in our animal experiment. Biochemical indicators of fecal, serum and liver were tested by kits or chemical methods. We also conducted the cholesterol micellar solubility experiment in vitro. Results: After 28 days of treatment, our results showed that WGP significantly reduced the serum levels of total cholesterol ( Triticum aestivum L.) is one of the three most common grains and is widely planted worldwide. The caryopsis of wheat is a staple food for human beings. After being milled into flour, the caryopsis can be used to make bread, steamed bread, biscuits, noodles and other foods [Wheat . The liver relative weight of the NC group was significantly lower than that of the HF, WG and WGP groups (p < 0.01). Therefore, by comparing with NC group, a high-fat diet alters the growth of rats and the WG and WGP treatment do not alter the growth of high-fat diet-induced rats. As shown in p < 0.05). No significant difference was observed in TG between the NC group, HF group, WG group and WGP group. No significant difference was observed in HDL-c between the NC group, HF group, WG group and WGP group. WGP intake significantly lowered non-HDL-c levels in the high-fat-diet rats compared with the HF group (p < 0.05). Under the treatment of WGP, the levels of TC and non-HDL-c returned to a normal level.As shown in p < 0.001). The administration of WG and WGP did not significantly affect the content of TC, TG or total lipids in rat liver compared with the HF group. However, after treatment with WG and WGP, the level of TC was found to be slightly higher than that in the HF group.As shown in p < 0.01). However, the concentration of HMG-CoA reductase in the WG and WGP groups was not significantly higher than that in the HF and NC groups. Moreover, the WG and WGP treatments also significantly increased the concentration of LDL-R compared with the HF and NC groups (p < 0.01). WG showed better results than WGP (p < 0.01). In comparison with the NC group, the high-fat diet cannot affect the enzyme activity of CYP7A1, HMG-CoA and LDL-R.As shown in p < 0.05) than in the HF group. Moreover, fecal excretion was significantly higher in the NC group than the HF, WG and WGP groups (p < 0.01). As shown in p < 0.01) and HF groups (p < 0.05).As shown in p < 0.001). Treatment with WGP may have a better effect in terms of inhibiting cholesterol absorption.As shown in p < 0.01), T-AOC, and CAT and reduced the level of serum MDA (p < 0.01) in high-fat diet-induced rats after 4 weeks compared with the HF group. The treatment of WG and WGP significantly improved the activity of serum SOD (p < 0.01), but the WG group was similar to the NC group. The levels of serum MDA in the WG and WGP groups were significantly higher than that of NC group (p < 0.01). However, treatment with either WG or WGP did not significantly change the activity of serum GSH-Px (erum SOD A and T-Aerum SOD B comparem GSH-Px C comparep < 0.05), and the administration of WGP induced a significant effect on T-AOC compared with the HF group (p < 0.05). For the level of GSH-Px, the HF, WG and WGP groups were significantly higher than NC group (p < 0.05), but no significant difference was observed in the HF, WG and WGP groups. The activity of SOD was significantly increased under the treatment of both WG and WGP (p < 0.01) compared with the NC group and HF group. Only WG significantly increased the activity of CAT (p < 0.05) compared with the NC group and HF group. The level of CAT in WGP group was significantly higher than in the NC group (p < 0.01) and similar with the HF and WG groups. The level of MDA in the liver decreased significantly with the WGP or WG treatment (p < 0.05) compared with HF. However, the level of MDA in WGP group was significantly higher than in the NC group (p < 0.01). For the liver antioxidant index, the administration of WG showed a better effect.It is well known that the increased level of serum cholesterol or lipid profiles can promote atherosclerosis and other cardiovascular diseases . An exceIn our research, we explored the hypolipidemic effect of wheat germ and wheat germ protein on high-fat-diet rats in vivo and in vitro. The administration of WG and WGP can effectively prevent metabolic complications in high-fat-diet rats with improvements in fecal and fecal bile acid excretion, a reduction in serum and liver lipid profiles, an increase in the protein levels involved in cholesterol metabolism, and modulate the antioxidant protein levels of serum and liver. In particular, we evaluated the inhibitory effect of WG and WGP on cholesterol micellar solubility. p < 0.05). Furthermore, WGP significantly promoted the excretion of fecal bile acids (p < 0.05). The classic bile acid’s biosynthetic pathway is generated by CYP7A1 by converting cholesterol to bile acid in the liver [In previous studies, a significant negative correlation was found between fecal steroid excretion and serum total cholesterol in rats . In our he liver . Thus, tp < 0.01). This result indicates that WG and WGP can effectively inhibit the absorption of cholesterol in intestinal. For better illustrating the cholesterol-lowing effect of WG and WGP in the intestines, we will explore the bile acid-binding ability and cholesterol absorption in Caco-2 cells in a future study.Many studies have stated that plasma cholesterol levels can be affected by dietary lipids . The uptp < 0.05). In a previous study, the treatment of wheat germ oil to hypercholesterolemic rabbits caused a significant reduction in serum cholesterol and non-HDL-cholesterol, in agreement with our results [Moreover, we discovered that the administration of WG and WGP results in significant reductions in serum total cholesterol (TC) and non-HDL-cholesterol concentrations in hypercholesterolemic rats fed a high-cholesterol diet . This result accords with our fecal experiments. These findings suggested that WG and WGP may enhance the conversion of cholesterol into bile acids and fecal excretion to promote the circulation of cholesterol. Cholesterol biosynthesis is a complex process involving more than 30 enzymes, and HMG-CoA reductase as a rate-limiting enzyme plays a pivotal role in the cholesterol biosynthetic pathway. So, inhibiting the activity and concentration of HMG-CoA reductase is one possible method to reduce cholesterol biosynthesis [p < 0.01). This result means that hepatocytes would uptake more LDL-c in serum than the high-fat diet group. Additionally, this result also accords with previous data that show that total cholesterol in serum decreased significantly (p < 0.05). These findings suggested that WG and WGP could lower the serum lipids by increasing the content of LDL-R. CYP7A1 as a rate-limiting enzyme regulates the cholesterol conversion to bile acid in the liver . Bile acynthesis . In our ynthesis ,33. Our 2O2. GSH-Px is an endogenous antioxidant, which can decompose peroxides by converting H2O2 into H2O and CO2 [2O2 and in the adaptive response to proton donors (ROOH). CAT somehow assists GSH-Px by converting H2O2 and ROOH into H2O [p < 0.01). WG also has a significant effect on increasing the content of CAT in the liver (p < 0.05), while WGP significantly decreased the concentration of MDA the in liver (p < 0.05). For serum, the SOD, T-AOC and CAT levels of WG and WGP groups had a significantly improvement compared with the HF group (p < 0.05). Furthermore, the MDA level in serum was significantly reduced under the treatment of WG and WGP (p < 0.05). These results indicated that WG and WGP are potential biosubstances that enhance the body’s antioxidant capacity. Moreover, WGP showed a better antioxidant capacity in serum.Many studies have clearly reported that high-fat diets can produce a great amount of free radicals and when their production exceeds the self-scavenging ability . Due to  and CO2 . Thus, Ginto H2O . The suminto H2O , etc. InTriticum aestivum L.) germ was purchased from the Inner Mongolia Hengfeng Food Industry (Group) Co., Ltd.  The wheat germ was cleaned and then turned into powder by passing it through a 60-mesh sieve. WGP was extracted from the wheat germ by alkali solution and acid isolation methods and then milled into powder. Wheat germ was dissolved in distilled water in a 2 L beaker to create a 10% solution, and then stirred for 2 h at 350 r/m at 50 ℃ with a constant temperature magnetic stirrer . The pH value was adjusted to 9.5 by 0.1 mol/L NaOH, and then, the solution was centrifuged at 3000× g for 20 min to obtain the supernatant. The pH of the supernatant was adjusted to pH 4.5 with 0.1 mol/L HCl, incubated for 2 h, and then centrifuged at 3000× g for 20 min to obtain the precipitate. The precipitate was washed with distilled water, and then the pH value was adjusted to pH 7.0. Finally, the residue was dried with a vacuum freeze dryer . The purity of the WGP was 80.10%.Wheat  Biotechnology Co. Ltd. The rats were housed in individual cages under a 12 h light–12 h dark cycle under temperature-controlled conditions at 22 ± 2 °C, with free access to food and water. After one week of being fed a commercial unpurified diet, the rats were divided into four groups. Animal procedures were approved by the Ethics Committee for Animal Research of the Hetao College .n = 5 in each group) based on body weight: normal control (NC) group, high-fat diet (HF) group, WG group and WGP group. The amino acid compositions of the WG and WGP are shown in g for 15 min at 4 °C, and then stored at −80 °C. The liver was collected, weighed and stored at −80 °C for further study.Finally, twenty rats were divided into four experimental groups  reductase, cholesterol 7-α hydroxylase (CYP7A1) activity and low-density lipoprotein receptor (LDL-R) in the liver were measured with Rat ELISA Kits . Total lipids of the liver were extracted by chloroform/methanol solution , as described by Folch et al. . The liv2SO4, 1 mL of 0.3% furfural was added after two minutes, and after 30 min of water bath at 65 °C the absorbance at 620 nm was recorded when the pink color developed with maximum intensity.The serum concentrations of TC, TG and high-density lipoprotein cholesterol (HDL-c) were enzymatically measured with commercial assay kits . TC extracted from fecal matter was measured with commercial assay kits . According to the method reported by Bruusgaard et al.  and MalcWe modified the cholesterol micelle method reported by Kirana et al. slightly . Lipids The total antioxidant capability (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), and malondialdehyde (MDA) of serum and liver were measured with assay kits .p < 0.05 was deemed to be a significant difference.All experimental dates are expressed as the means ± SEMs (standard error of mean). The data of the rats were analyzed by using SPSS software version 21. Data were analyzed by one-way analysis of variance (ANOVA), followed by LSD (L) test and p < 0.05). Such benefits were associated with the WGP-mediated downregulation of cholesterol micellar solubility and upregulation of enzymes involved in cholesterol metabolism, including CYP7A1 and LDL-R. Furthermore, WGP significantly decreased the content of MDA and increased the activity of SOD, CAT and the capacity of T-AOC (p < 0.05). However, WG did not significantly increase bile acid excretion in feces and decrease the serum levels of TC. These findings indicated that WGP may represent a viable, safe and attractive plant protein approach to attenuate the hyperlipidemia.In summary, we investigated the hypolipidemic effect of WG and WGP from wheat in high-fat diet-induced rats. The treatment of WGP significantly increased the concentration of fecal bile acids and decreased the levels of TC and non-HDL-c in serum (


Time to diabetes was similar in the two cohorts . At multivariable regression analysis, incident diabetes was significantly associated with prediabetes (odds ratio (OR) 4.40; 95% confidence interval (CI) 1.97–9.81; p < 0.001), percent weight change  and higher education , with no effect of other baseline socio-demographic, behavioral and clinical data, and of the type of intervention. The importance of weight change on incident diabetes were confirmed in a sensitivity analysis limited to individuals who completed the follow-up. Conclusion: In individuals with NAFLD, WBI is as effective as GBI on the pending long-term risk of diabetes, via similar results on weight change. Background: Behavioral programs are needed for prevention and treatment of NAFLD and the effectiveness of a web-based intervention (WBI) is similar to a standard group-based intervention (GBI) on liver disease biomarkers. Objective: We aimed to test the long-term effectiveness of both programs on diabetes incidence, a common outcome in NAFLD progression. Methods: 546 NAFLD individuals  were followed up to 60 months with regular 6- to 12-month hospital visits. The two cohorts differed in several socio-demographic and clinical data. In the course of the years, the average BMI similarly decreased in both cohorts, by 5% or more in 24.4% and by 10% or more in 16.5% of cases available at follow-up. After excluding 183 cases with diabetes at entry, diabetes was newly diagnosed in 48 cases during follow-up (31 (16.6% of cases without diabetes at entry) in the GBI cohort vs. 17 (9.7%) in WBI;  Nonalcoholic fatty liver disease (NAFLD) is one of the most common and rapidly growing conditions in the world, affecting approximately 25% of the total adult population , and an To facilitate adherence, on-line and off-line web-based programs , as wellAs part of a European program, we developed a web-based behavioral program mimicking all the activities of the group-based obesity classes. The aim was to eliminate space and time constraints that limit attendance to busy liver units, to spare patients’ and physicians’ time, thus possibly expanding lifestyle intervention to a much larger community. The two-year results have been published in 2018, supporting the use of the web-based intervention as possible alternative to hospital attendance .Because of the association with other non-communicable diseases, particularly obesity and type 2 diabetes, progression from fatty liver to nonalcoholic steatohepatitis (NASH), to fibrosis, cirrhosis and to end-stage liver disease, also carries a relevant risk of extra-hepatic comorbidities. Cardiovascular  and renaAs part of the long-term surveillance of our NAFLD cohort, the aim of the present report is to provide a comparative analysis of diabetes incidence in patients enrolled in either the group-based or the web-based educational intervention during a 5-year real-world follow-up.The original study involved individuals with ultrasonography-diagnosed NAFLD attending the Unit of Metabolic Diseases and Clinical Dietetics, University of Bologna, from January 2012 to December 2015, as well as a small group of individuals who were enrolled into the web-based program by the Department of Gastroenterology, University of Turin. The protocol of the educational intervention, funded as part of an EU subproject FP7/2007- 2013 FLIP (Fatty Liver—Inhibition to Progression), under grant agreement No. HEALTH-F2-2009-241762, has been previously published . AccordiThis long-term (5-year) follow-up was limited to 546 subjects, who completed either program, attended the six-month control visit, and were followed until December 2019, when the SARS-CoV-2 pandemic caused a disruption of hospital procedures; their baseline data are reported in The study was initially approved by the ethical committee of Sant’Orsola-Malpighi Hospital, Bologna, as an interventional, non-pharmacologic study (No. 79/2009/U/Oss), and patients signed an informed consent before entering the program. Long-term comparison with the standard treatment (GBI) is part of an internal audit to test the effectiveness of WBI on specific outcomes. The primary outcome of the present analysis was diabetes incidence. Secondary outcomes were glucose and HbA1c control in subjects with type 2 diabetes at enrollment. The occurrence of diabetes was defined by fasting glucose levels exceeding the threshold of 126 mg/dL or by HbA1c levels ≥ 6.5% (48 mmol/mol) or by the ICD-9 code 250.0 during hospital admission and/or glucose-lowering treatment at follow-up. Prediabetes was defined by the presence of fasting glucose between 100 and 125 mg/dL or HbA1c between 5.7 and 6.4% (39–47 mmol/mol) or impaired glucose tolerance .Calorie intake, both at entry and at the end of the educational intervention (after 4–6 months in both cohorts), was semi-quantitatively assessed by an in-house developed self-administered questionnaire, available either during the six-month control visit or on line. The questionnaire is based on the weekly consumption and portion size  of 18 items related to habitual food intake, and a final item on the number of meals not consumed at home during the week (to account for the possible extra food intake when eating at restaurant). To help subjects with portion size, pictures are included to visually explain what is considered small-sized, medium- sized, or large-sized, whereas a few questions specifically investigate the number of certain items consumed during an average week  . The queHabitual physical activity was measured at the same time points by the International Physical Activity questionnaire .Cigarette smoking was classified as active, previous, and never smoking. Safe limits of alcohol intake in non-abstainers were set as ≤14 units per week in females, ≤21 in males. n = 363) and a drop-out rate of 30%, in a 5-year follow-up the expected number of cases with incident diabetes was 73. Under these assumptions, the sample size was considered sufficiently powered to test the effectiveness of the intervention with an α-error of 0.05 and a β-error of 0.20.In the calculation of sample size, giving the high prevalence of prediabetes at entry and the high risk associated with NAFLD, the risk of diabetes at follow-up might be estimated at 8.0 per 100 patient-years , expectet test for unpaired data, chi-square test or Mann-Whitney rank test, whenever appropriate. Time x treatment ANOVA was used to test differences in the time-course of individual parameters between groups in subjects retained in follow-up. The cumulative risk and the relative risk of incident diabetes in the GBI- and WBI-based cohorts were determined by Kaplan-Meier and Cox proportional hazard analysis, respectively. Factors associated with primary and secondary outcomes (dependent variable) were tested by logistic regression analysis, having type of intervention and confounders as independent variables. Given the importance of weight loss as factor protecting from diabetes and improving metabolic control, diabetes risk was corrected for percent weight change between enrollment and time of diabetes incidence or end of follow-up; metabolic control and glucose target reach at any time point were also corrected for percent weight change. Descriptive statistics were made by computing means ± standard deviation for the entire, the WBI and the GBI cohorts. For nominal data, the prevalence and the 95% confidence interval were calculated. Comparison between groups was carried out by Student 2: 12.0% vs. 15.7%, respectively)—in the two cohorts.Remarkable differences between GBI and WBI cohorts were present at enrollment . The GBIp = 0.005, Mann-Whitney rank test). Attrition rates were high  and higher in WBI vs. GBI .The length of follow-up averaged 41.1 ± 22.7 months and was significantly shorter in the WBI cohort , without differences between groups. Physical activity was generally low, but significantly higher in the WBI cohort. By the end of the educational period, it had increased in both cohorts to 26.6 ± 14.1 MET/hour/week (WBI-treated) and to 22.2 ± 16.3 (GBI-treated), and the differences between the two groups remained statistically significant.Smoking and alcohol habits were similar. Daily calorie intake was also similar in the two groups at entry. Following the educational intervention, it decreased by 194 ± 286 kcal/day in GBI and by 233 ± 334 kcal/day in WBI, to final values of 1717 ± 276 kcal/day and 1692 ± 247, respectively (p < 0.001) and 127 more were diagnosed as prediabetes . Prediabetes accounted for 41.7% of GBI- and 27.8% of WBI-treated cases without overt diabetes. Glycosylated hemoglobin (HbA1c) was on average 7.58 ± 1.45% in subjects with diabetes vs. 5.66 ± 0.60 in subjects without diabetes.At entry, diabetes was recorded in 183 cases (147 (44.0%) GBI- and 36 (17.0%) WBI-treated cases; p < 0.001), whereas it increased in subjects without diabetes at entry (by 0.18 ± 0.71%), given newly-diagnosed diabetes.During follow-up, HbA1c declined by only 0.11 ± 1.08% in the whole cohort, but it was remarkably reduced in the population with diabetes at entry . Five cases were diagnosed after six months, 11 after one year, and then 6, 12, 8 and 6 after two, three, four and five years, respectively. Time to diabetes was similar in the two cohorts (on average 31 ± 18 months after enrollment (range 6–60 months). In the newly-detected diabetes subgroup, HbA1c at enrollment was 5.89 ± 0.75% and increased by 0.82 ± 0.91% during follow-up to final values of 6.79 ± 0.86% at last observation, despite antidiabetic treatment.Incident diabetes was indeed observed in 48 cases (31 (16.6% of cases without diabetes at entry of the GBI cohort); 17 (9.7%) in WBI; p = 0.084), whereas it remained relatively stable in 23.6%, decreased by 5% or more in 24.4% and by 10% or more in 16.5%. Overall, time x treatment ANOVA revealed differences in weight trajectories between the two interventions in favor of the WBI-based cohort , largely driven by the reduced number of cases who increased their body weight by the end of follow-up in the GBI cohort. In response to the intervention, the average BMI was reduced by approximately 4%, without differences between groups, and remined relatively stable, until year 4 and 5, when a progressively larger variability was observed . This wan = 48, −0.3 ± 8.0% vs. −4.4 ± 6.0% in 315 cases who did not develop diabetes; p < 0.001) (The average percent weight change was significantly different in relation to incident diabetes (Incident diabetes: < 0.001) .The cumulative incidence of diabetes was moderately higher in the GBI-based cohort, also driven by the higher rate of prediabetes. However, Kaplan-Meier analysis  failed tn = 179; incident diabetes, n = 41); percent weight change was again negatively associated with newly-detected diabetes . By univariate logistic regression analysis , severalAlso in subjects with diabetes at entry HbA1c more markedly decreased in those who achieved a significant weight loss (on average −0.61 ± 1.28% in subjects who attained a weight loss of 5% or more and −0.77 ± 1.48% for a weight loss exceeding 10%), independently of the type of treatment. However, in these groups also the pharmacologic treatment of diabetes was changed in the course of the years in order to attain a better metabolic control, as suggested by most recent guidelines, and the relative importance of weight loss could not be tested. The report shows that an intensive lifestyle intervention, carried out either in groups or delivered by web in motivated individuals with NAFLD reduces the long-term risk of incident diabetes and, in subjects with diabetes, it has a beneficial effect on metabolic control. The effect is strictly dependent on the baseline impairment in glucose metabolism, but also on the amount of weight loss achieved during follow-up, that was remarkably similar in the two cohorts treated by the GBI- and the WBI-based programs.Type 2 diabetes and obesity are the leading factors associated with NAFLD , and theThe link between NAFLD and diabetes incidence is well established. Even modestly increased levels of alanine amino-transferase or gamma-glutamyl-transpeptidase are associated with risk of diabetes incidence ,30,31,32The effectiveness of lifestyle intervention in the prevention of diabetes was definitively established in three seminal studies in subjects with prediabetes: the Diabetes Prevention Program , the Dian = 363). Prediabetes and weight loss are the factors more closely associated with incident diabetes , independently of treatment program. This confirms that an effective educational treatment may be delivered via internet, saving time and cost for both patients and health care facilities. An old analysis showed that the effect sizes of a few treatment outcomes, including increased exercise time and participation in healthcare programs, as well as weight loss maintenance, were even better addressed by web-based interventions [The basic strategies of lifestyle intervention have later been applied in several settings and different countries, confirming that healthy lifestyle driving weight loss is the cornerstone of diabetes prevention ,41,42,43ventions  in compaIn addition to liver-related events, cardiovascular outcomes and chronic kidney disease (CKD), as well as hepatic and extra-hepatic cancers, are common outcomes of NAFLD , also shLimitations should however be considered. First, our WBI program was not entirely internet-based. Although patients could interact with the center off-line and could send food diaries or ask questions via dedicated tools, this was usually limited to the first few months from enrollment. In the long-term, the initial motivational visit and the continuous support provided by reinforcements at follow-up visits, similar to that offered to the GBI cohort, could also have played an important role. Second, the two cohorts were markedly different at entry in their socio-demographic and clinical parameters. We used a large set of possible confounders to adjust the final outcomes, but the possibility remains that the results of the WBI approach might differ from those attained by the GBI approach. The WBI approach was prefered by subjects who had higher education—suggesting higher health literacy, younger age, more active job involvement, that could reduce attendance to the fixed and multiple sessions of the WBI approach. On the contrary, the GBI cohort included individuals that might have difficulties interacting with technology. Thus, the two programs target different populations that could hardly be compared in randomized studies.In addition, the WBI strategy was biased by a larger attrition rate, also resulting in a shorter length of follow-up. The high attrition rate is comparable to that observed in most behavioral intervention studies ,49,50, lThird, it should be noted that the putative effects of behavioral treatment  were not sequentially measured at follow-up. However, weight loss and weight loss maintenance might be considered a surrogate biomarker of healthy lifestyle adherence. Future studies should consider sequential measurement of habitual physical activity and dietary intake. Patients of both cohorts were trained to adhere to the principles of Mediterranean diet, that has been associated with both weight loss, steatosis regression and better cardiovascular risk profile in NAFLD . HealthyIn conclusion, the report provides evidence that a web-based educational intervention is as effective as a behavioral intervention based on group sessions in promoting weight changes that negatively associate with long-term diabetes risk in NAFLD individuals. From a health-care viewpoint, the larger use of web-based approaches is likely to extend behavioral treatment to a broader audience, thus reducing healthcare costs in the present pandemic of non-communicable diseases.